The present invention relates to methods and compositions for preventing or treating mycobacterium infections, particularly tuberculosis infections.
Infectious diseases remain the largest cause of death in the world today, greater than cardiovascular disease or cancer.1 Among infectious diseases, tuberculosis (TB) is the leading cause of death.2 
Tuberculosis, caused by the infection of Mycobacterium tuberculosis (Mtb), kills three million people worldwide and eight million people develop the disease each year according to current estimates by the World Health Organization (WHO). More people die from TB than from malaria, diarrhea, AIDS and tropical diseases combined.
Tuberculosis mainly affects the lungs but can also involve other organs. TB strikes people of all ages, but is more common among the elderly. The disease can also afflict animals, especially livestock such as cattle, hogs and poultry. This disease once ranked among the most common causes of death in the world. Today, improved methods of prevention, detection, diagnosis and treatment have greatly reduced both the number of people who contract the disease and the number of people who die from it. However, in the last decade, the outbreaks of multidrug-resistant tuberculosis (MDRTB) and TB amplified by the global HIV pandemic make TB an urgent global issue.
One third of the world""s population is infected with Mtb,3 a facultative intracellular bacillus. After infection with Mtb, the lifetime risk of developing TB is approximately 10%, while 90% of infected persons have latent infection with viable bacilli. This 10% rate of TB accounts for the 8 million persons reported annually with active TB, and the resultant 3 million deaths. Moreover, TB is a serious problem faced by hemodialysis patients,4 and is the number one killer of women of childbearing age around the world, with 1.2 million women dying of the disease in 1997, according to reports by the WHO.5 
TB infection is a serious problem for acquired immunodeficiency syndrome (AIDS) patients. HIV-infected individuals are particularly susceptible to infection with Mtb and the development of TB. Compared to an individual who is not infected with HIV, an individual infected with HIV has a 10 times greater risk of developing TB. In an individual infected with HIV, the presence of other infections, including TB, may allow HIV to multiply more quickly. This may result in more rapid progression of HIV infection and AIDS.6 As HIV infection progresses, CD4+ lymphocytes decline in number and function. The immune system is less able to prevent the growth and local spread of Mtb. Even in HIV-infected patients, pulmonary TB (PTB) is still the most common form of TB. The presentation of the disease depends on the degree of immunosuppression. As in adults, the natural history of TB in a child infected with HIV depends on the stage of HIV disease. Early in HIV infection, when immunity is strong, the signs of TB are similar to those in a child without HIV infection. As HIV infection progresses and immunity declines, dissemination of TB becomes more common and tuberculous meningitis, miliary tuberculosis, and widespread tuberculous lymphadenopathy occur more frequently.
HIV-positive patients and staff in health units face daily exposure to TB. The risk of exposure is greatest in adult medical wards and TB wards where there are many PTB cases. From 1990-1992, the Centers for Disease Control (CDC) investigated outbreaks of MDRTB in several hospitals and a state correctional system. Almost 300 cases of MDRTB were identified in these outbreaks; most patients were HIV-seropositive. The mortality rate was 80%-90% and the median interval from diagnosis of tuberculosis to death ranged from 4-16 weeks.7 In 1995, about one third of the 17 million HIV-infected people worldwide were also co-infected with Mtb.6 
Current treatment of TB requires taking at least two antibiotics, usually isoniazid and rifampicin, supplemented with pyrazinamide and ethambutol added when isoniazid resistance is suspected.
Isoniazid (isonicotinic acid hydrazide) (INH) was first reported to be effective against Mtb and M. bovis in 1952.8-10 Isoniazid, now still a front-line therapy against TB, has been shown to be an effective prophylactic antitubercular11, and modern short-course chemotherapy is initiated with three drugs: isoniazid, rifampin and pyrazinamide (PZA), often with the inclusion of a fourth drug, usually ethambutol. Recently, rifapentine, a derivative of rifamycin, was approved by the FDA for the treatment of tuberculosis.12 
The American Thoracic Society and the CDC in the United States now recommend a treatment regimen of isoniazid, rifampin and pyrazinamide for 2 months, followed by isoniazid and rifampin for an additional 4 months, as the standard 6-month regimen. Isoniazid, cheap and safe, has a wide therapeutic margin and high early bactericidal activity so that it kills rapidly growing bacilli in lesions, but is inefficient in ultimately sterilizing these lesions. Rifampin and PZA are crucial in achieving sterilization by killing persisting semi-dormant bacilli, and are thus responsible for shortening the duration of treatment from the earlier norm of 12-18 months to the current standard of 6 months.13 However, many people fail to complete the lengthy therapy, treatment failures are high and MDR is increasing. A four-year study, led by the WHO, shows that of people who had been treated for TB for less than a month, 36 percent harbored microbes that resisted at least one of the four main anti-tuberculosis drugs. Moreover, 10% of infected people who had never been treated for the disease carried a strain of Mtb that resisted at least one drug.14 
Drug resistance resulting from inadequate treatment, such as irregular drug supply, inappropriate regimens or poor compliance is a potential threat to TB control programs throughout the world. Patients infected with strains resistant to multiple drugs are less likely to be cured, particularly if they are infected with HIV or malnourished, and their treatment is more toxic and more expensive than the treatment of patients with susceptible organisms.
The resurgence of TB, the development of MDR to Mtb and the discovery that the progression of TB is accelerated in HIV-positive patients have intensified the need to develop more efficient drugs to combat this disease.
The present invention relates to compounds, compositions and methods for treating and/or preventing mycobacterium infections, especially tuberculosis infections, in patients. The method is useful for treating or preventing mycobacterium infections in immunocomprised patients, particularly HIV infected patients.
The present invention relates to compounds, compositions and methods for the prevention or treatment of mycobacterium infections. The compounds are naturally occurring and synthetic biflavonoids, flavonoids, chalcones and chalcone like compounds. The compounds were screened for anti-mycobacterium activity and several were found to cause inhibition of a mycobacterium infection. Of these, eight were identified as particularly potent, exhibiting greater than 90% inhibition of the growth of Mtb at a concentration of 12.5 xcexcg/mL. The actual minimum inhibitory concentrations (MIC), defined as the lowest concentration inhibiting 99% of the inoculum, for the preferred compounds ranged from 6.8 to 48.3 xcexcM.
Accordingly, one object of the invention is a method for preventing or treating a mycobacterium infection in a mammal comprising administering to a mammal in need of anti-mycobacterium prevention or treatment an effective anti-mycobacterium amount of at least one compound of formula i. 
wherein R1-R9 are independently comprised of H; OCH3; EtO; OH; O-alkenyl; phenyl; NH2; COOH; F; Cl; Br; I; CONH2; NO2; NR10R11 OCONR10R11 wherein R10 and R11 independently comprise H alkyl (e.g., C1-6 linear or branched alkyl) or aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen); COR12 wherein R12 comprises H, OH, O-alkyl (e.g., C1-6 linear or branched alkyl), O-aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen) or amino; NHCOCH3; O2xe2x88x92; OCOR13 wherein R13 comprises alkyl (e.g., C1-6 linear or branched alkyl) or aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen); OAc; benzoyl; CONH2; or NO2; or a pharmaceutically acceptable derivative or salt thereof.
Compounds of the formula i as well as anti-mycobacterium compositions comprising compounds of the formula i are included within this aspect of the invention.
Another object of the invention is a method for preventing or treating a mycobacterium infection in a mammal comprising administering to a mammal in need of anti-mycobacterium prevention or treatment an effective anti-mycobacterium amount of at least one compound of formula ii. 
wherein R1 comprises 4-fluorophenyl-, 3-hydroxyphenyl-, pyridin-3-yl-, furan-2yl-, phenanthren-2-yl-, 3-fluorenyl-, pyridin-2-yl-, naphthalen-1-yl-, pyridin-2-yl-, 4-bromo-2-hydroxyphenyl-, pyridin-4-yl-, 2-hydroxy-4-methoxyphenyl-, 4-aminophenyl-, pyridin-4-yl-, 2-hydroxy-5-methoxyphenyl-, 4-methoxyphenyl-, 4-methoxyphenyl-, 2-hydroxy-5-chlorophenyl-, 4-aminophenyl-, 3-hydroxynaphthalen-2-yl-, furan-2-yl- or pyridin-2-yl-; and
R2 comprises pyridin-3-yl-, phenanthren-9-yl-, phenanthren-9-yl-, phenyl-, 2-aminopyridino-3-yl, 2-aminopyridino-3-yl-, pyridin-2-yl-, phenyl-, 4-dimethylaminophenyl-, furan-2-yl-, indol-2-yl-, furan-2-yl-, 2-aminopyridin-3-yl-, 4-dimethylaminophenyl-, furan-2-yl-, pyridin-4-yl-, pyridin-3-yl-, 2-aminopyridin-3-yl-, 2-aminopyridin-3-yl-, 2-aminopyridin-3-yl-, pyridin-4-yl- or 4-methoxyphenyl-; or a pharmaceutically acceptable derivative or salt thereof.
Compounds of the formula ii as well as anti-mycobacterium compositions comprising compounds of the formula ii are included within this aspect of the invention.
Another object of the invention is a method for preventing or treating a mycobacterium infection in a mammal comprising administering to a mammal in need of anti-mycobacterium prevention or treatment an effective anti-mycobacterium amount of at least one compound of formula iii. 
wherein R1-R8 are independently comprised of H; OCH3; EtO; OH; O-alkenyl; sdphenyl; NH2; COOH; F; Cl; Br; I; CONH2; NO2; NR10R11 OCONR10OR11 wherein R10 and R11 independently comprise H alkyl (e.g., C1-6 linear or branched alkyl) or aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen); COR12 wherein R12 comprises H, OH, O-alkyl (e.g., C1-6 linear or branched alkyl), O-aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen) or amino; NHCOCH3; O2xe2x88x92; OCOR13 wherein R13 comprises alkyl (e.g., C1-6 linear or branched alkyl) or aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen); OAc; benzoyl; CONH2; or NO2; or pharmaceutically acceptable derivative or salt thereof.
Compounds of the formula iii as well as anti-mycobacterium compositions comprising compounds of the formula iii are included within this aspect of the invention.
Another object of the invention is a method for preventing or treating a mycobacterium infection in a mammal comprising administering to a mammal in need of anti-mycobacterium prevention or treatment an effective anti-mycobacterium amount of at least one compound of formula iv. 
wherein R1-R8 are independently comprised of H; OCH3; EtO; OH; O-alkenyl; phenyl; NH2; COOH; F; Cl; Br; I; CONH2; NO2; NR10R11 OCONR10R11 wherein R10 and R11 independently comprise H alkyl (e.g., C1-6 linear or branched alkyl) or aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen); COR12 wherein R12 comprises H, OH, O-alkyl (e.g., C1-6 linear or branched alkyl), O-aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen) or amino; NHCOCH3; O2xe2x88x92; OCOR13 wherein R13 comprises alkyl (e.g., C1-6 linear or branched alkyl) or aryl (e.g., unsubstituted phenyl or phenyl substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, hydroxy-C1-4 alkyl, hydroxyl, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, amino-C1-8 alkyl, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, nitro, azido or halogen); OAc; benzoyl; CONH2; or NO2; or pharmaceutically acceptable derivative or salt thereof.
Compounds of the formula iv as well as anti-mycobacterium compositions comprising compounds of the formula iv are included within this aspect of the invention.
Yet another object of the invention is a method for treating or preventing mycobacterium infection in a patient comprising biflavonoid compounds, particularly 6-6xe2x80x3-biapigenin hexamethylether, volkensiflavone hexamethylether, GB-1a hexamethylether, 3xe2x80x3xe2x80x2-Nitro-C3-O-C4xe2x80x3xe2x80x2-biflavone, 3xe2x80x2-8xe2x80x3-biflavone, 6-2xe2x80x3xe2x80x2-biflavone, 6-6xe2x80x3-binaringenin hexamethyl-ether or 6-2xe2x80x3xe2x80x2-biapigenin or derivative or salt thereof and pharmaceutically acceptable carriers therefor.
Still yet another object of the invention is to provide anti-mycobacterium composition comprising biflavonoid compounds for treating or preventing a mycobacterium infection in a patient particularly 6-6xe2x80x3-biagpigenin hexamethylether, volkensiflavone hexamethylether, GB-1a hexamethylether, 3xe2x80x3xe2x80x2-Nitro-C3-O-C4xe2x80x3xe2x80x2-biflavone, 3xe2x80x2-8xe2x80x3-biflavone, 6-2xe2x80x3xe2x80x2-biflavone, 6-6xe2x80x3-binaringenin hexamethylether or 6-2xe2x80x3xe2x80x2-biapigenin or derivative or salt thereof and pharmaceutically acceptable carriers therefor.
These and other objects of the invention will be clear in light of the detailed description below.
This invention relates to compounds, compositions and methods for treating or preventing mycobacterium infections in mammals. The compounds of the present invention are synthetic or naturally occurring chalcones, chalcone-like compounds, biflavonoids and flavonoids. The compounds were screened for anti-mycobacterium activity. Of the compounds showing anti-mycobacterium activity, eight were identified as particularly potent, exhibiting greater than 90% inhibition of the growth of Mtb at a concentration of 12.5 xcexcg/mL.
The preferred compounds of this invention, which exhibited greater than 90% inhibition of the growth Mtb at a concentration of 12.5 xcexcg/mL, were chalcone-like compounds (heterocyclic ring substituted 2-propen-1-one) 1-(4-fluorophenyl)-3-(pyridin-3-yl)-2-propen-1-one (53) (98%), 1-(3-hydroxyphenyl)-3-phenanthren-9-yl-2-propen-one (54) (97%), 1-(5-pyridin-2-yl)-3-(phenanthen-9-yl)-2-propen-1-one (55) (96) and 1-(furan-2-yl)-3-phenyl-2-propen- 1-one (56) (96%); chalcones 1-(2-hydroxyphenyl)-3-(3-chlorophenyl)-2-propen-1-one (24) (90%) and 1-(2-hydroxyphenyl)-3-(3-iodophenyl)-2-propen-1-one (40) (92%); and biflavonoids 6-6xe2x80x3-biapigenin hexamethylether (151) (96%), and volkensiflavone hexamethylether (3-8xe2x80x3-naringenylapigenin hexamethylether) (152) (95%). The actual minimum inhibitory concentrations (MIC), defined as the lowest concentration inhibiting 99% of the inoculum, for 53, 54, 55, 56, 24, 40, and 151 were 6.8, 19.2, 20.2, 31.5, 48.3,  greater than 35.7 and  greater than 20.1 xcexcM, respectively. See Tables 1-5.
The compounds and compositions of the present invention can be used to treat or prevent mycobacterium infections. Representative mycobacterial organisms include Mycobacterium aviurn complex (MAC), Mycobacterium kansaii, Mycobacterium marinum, Mycobacterium phlei, Mycobacterium ulcerans, Mycobacterium xenopi, Mycobacterium gordonae, Mycobacterium terrae complex, Mycobacterium haemophilum, Mycobacterium fortuitum, Mycobacterium tuberculosis, Mycobacterium laprae, Mycobacterium scrofulaceum and Mycobacterium smegmatis. In practicing this invention, the compounds and compositions are particularly useful in treating Mycobacterium tuberculosis infections.
The compounds of the invention may be formulated as a solution of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqeuous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or in buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium choride or sodium citrate.
Alternatively, the compounds of the present invention may be encapsulated, tableted or prepared in an emulsion (oil-in-water or water-in-oil) syrup for oral administration. Pharmaceutically acceptable solids or liquid carriers, which are generally known in the pharmaceutical formulary arts, may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch (corn or potato), lactose, calcium sulfate dihydrate, terra alba, croscarmellose sodium, magnesium stearate or stearic acid, talc, pectin, acacia, agar, gelatin, maltodextrins and microcrystalline cellulose, or colloidal silicon dioxide. Liquid carriers include syrup, peanut oil, olive oil, corn oil, sesame oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 10 mg to about 1 g per dosage unit.
The dosage ranges for administration of the compounds of the invention are those necessary to produce the desired affect whereby symptoms of infection are ameliorated. For example, as used herein, an anti-mycobacterium effective amount for treating or preventing a mycobacterium infection refers to the amount administered so as to maintain an amount which suppresses or inhibits mycobacterium infection as evidenced by standard assay. The dosage will also be determined by the existence of any adverse side effects that may accompany the compounds. It is always desirable, whenever possible, to keep adverse side effects to a minimum.
One skilled in the art can easily determine the appropriate dosage, schedule, and method of administration for the exact formulation of the composition being used in order to achieve the desired effective concentration in the individual patient. However, the dosage can vary from between about 0.001 mg/kg/day to about 50 mg/kg/day, but preferably between about 0.01 to about 1.0 mg/kg/day.